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Protocol

Solutions and buffers for ISH

Acc.no: 1VGYAYS | Published: 2010-03-23 by jennie

Keywords: ISH, in situ, hybridization, buffer

Solutions and buffers for In Situ Hybridization

Nitril gloves should always be worn when working with formamide. Work in biosafety cabinet and use sealed zip lock bags whenever you need to move your samples.

Required Materials

Main steps

  1. Proteinase K Stock Solution (5 mg/ml)
  2. NTMTw (1000ml)
  3. NBT/BCIP Solution
  4. Levamisole Solution
  5. NBT/BCIP and Levamisole Solution
  6. KTBTw stop solution (1000 ml)
  7. 1 M Tris pH 7.5 (1000 ml)
  8. 1 M Tris pH 9.5 (1000 ml)
  9. 0.5 M Tris pH 8.0 (250 ml)
  10. Yeast tRNA (25 mg/ml)
  11. Heparin 50 mg/ml
  12. Antibodies
  13. Blocking Solution 50m to be stored at 5˚ degrees
  14. Blocking Solution 50 ml
  15. 50 mM Tris pH 7.5 (50 ml)
  16. Proteinase K Working Solution (50 µg/ml)
  17. Acetic anhydride/triethanolamine
  18. 4% formaldehyde (200 ml)
  19. Prehybridization Buffer (50 ml)
  20. Hybridization Buffer
  21. Chamber Humidity Solution (500 ml)
  22. Washing buffer 1 (1000 ml)
  23. Washing buffer 2 (1000 ml)
  24. Washing buffer 3 (1000 ml)
  25. Troubleshooting

Instructions

  1. Proteinase K Stock Solution (5 mg/ml)

    Dissolve 5 mg /ml of Proteinase K in Molecular grade water. Stored at -20˚ C.

  2. Levamisole Solution

    Add levamisole to the NBT/BCIP to a final concentration of 2 mM. 0.258 g Levimisole Adjust to 10.75ml MilliQ H20

  3. NBT/BCIP and Levamisole Solution

    Add levamisole to the NBT/BCIP to a final concentration of 2 mM.

  4. KTBTw stop solution (1000 ml)

    50 ml Tris-HCl (pH 7.5 )(50 mM) 0.75 g KCl(10mM) 10 ml 100% Tween 20 (1%) 8.8 g NaCl (150 mM) Adjust to 1000 ml with MilliQ H20

  5. 1 M Tris pH 7.5 (1000 ml)

    Add 121 g Tris-Base to 700 ml H20 Adjust pH to 7.5 with concentrated HCL Adjust to 1000 ml with MilliQ H20

  6. 1 M Tris pH 9.5 (1000 ml)

    Add 121 g Tris-Base to 700 ml H20 Adjust pH to 9.5 with concentrated HCL Adjust to 1000 ml with MilliQ H20

  7. 0.5 M Tris pH 8.0 (250 ml)

    Add 15.1 g Tris-Base to 175 ml milliQ H20 Adjust pH to 8.0 with concentrated HCL Adjust to 250 ml with milliQ H20

  8. Yeast tRNA (25 mg/ml)

    Dissolve 250 mg yeast tRNA in 10 ml of DEPC (Molecular grade water).

  9. Heparin 50 mg/ml

    Dissolve 50 mg Heparin in 1 ml DEPC H20.

  10. Troubleshooting

    • Be careful when using PBS together with a AP based detection system as phosphate ions can inhibit the activity of the enzyme.

    • For DIG System applications do not include MgCl¬2 in the detection buffer as this might lead to spotty background on the slide after the detection procedure.

  11. Proteinase K Stock Solution (5 mg/ml)

    Dissolve 5 mg /ml of Proteinase K in Molecular grade water. Stored at -20˚ C.

  12. 50 mM Tris pH 7.5 (50 ml)

    Dilute 2.5 ml of 1M Tris pH 7.5 in 47.5 ml H2O in a 50 ml Nunc tube.

  13. Proteinase K Working Solution (50 µg/ml)

    Dilute the Proteinase stock solution 1µl Proteinase-K + 99 µl 50 mM Tris pH 7.5

  14. Acetic anhydride/triethanolamine

    2.33 ml triethanolamine, 500 µl acetic anhydride, add to 200 ml with DEPC (Molecular grade water).

    Use immediately!

  15. 4% formaldehyde (200 ml)

    22 ml 3 % formaldehyde (4%) Adjust to 200 ml with PBS DEPC (Molecular grade water).

  16. Prehybridization Buffer (50 ml)

    25.0 ml 99,9% formamid,(50%) 50 µl 50 mg/ml heparin (50 µg/ml) 100 µl 25 µg/ml yeast RNA (50 µg/ml 1.0 g Blocking Powder (2%) 50 µl 100 % tween 20 (0,1%) 12.5 ml 20xSSC (5x) 0.093 g EDTA (5 mM) Adjust to 50 ml with DEPC H20

    Store as aliquots (1 ml) at minus 80° C

  17. Hybridization Buffer

    Molecular mass NaCl 58.44 g/mol

    25.0 ml 99.9% formamid,(50%) 0.9 g Nacl (0.3M) 2.0 ml 0.5 M Tris-HCl pH 8.0 (20 mM) 5 g Dextran Sulphate (10%) 1 ml 50x Denhardts (1x) 1 ml 25 mg/ml yeast tRNA (0,5 mg/ml)

    Store as aliquots (1 ml) at minus 80° C

  18. Chamber Humidity Solution (500 ml)

    250 ml 99.9% formamide (50%) 25 ml 20x SSC (1x) Adjust to 500 ml with MilliQ H20

  19. Washing buffer 1 (1000 ml)

    500 ml 99,9% formamide (50%) 1,0 ml 100% Tween 20 (0,1%) 200 ml 5x SSC (1x) Adjust to 1000 ml with H20

  20. Washing buffer 2 (1000 ml)

    10 ml 20x SSC (0,2x) Adjust to 1000 ml with H20

  21. Washing buffer 3 (1000 ml)

    1 ml 100% Tween 20 (0.1% final) Adjust to 1000 ml with PBS

  22. Blocking Solution 50 ml

    0,25 g blocking powder(0,5%) 5 ml heatinactivated goat serum (10%) 50 µl 100% Tween 20(0.1%) Adjust to 50 ml with PBS

    Store aliquots (1000ul) at -20° C

    Heat blocking powder at 50° C for a few hours or O/N

  23. Blocking Solution 50m to be stored at 5˚ degrees

    0,25g blocking powder (0.5%) Adjust to 50 ml with PBS

    Heat blocking powder at 50° C for a few hours or O/N

  24. Antibodies

    1:250 or 1:500 anti DIG-AP Fab fragments (Roche 109327) in 0.5 % blocking buffer stored at 5˚ C.

  25. NTMTw (1000ml)

    Molecular mass NaCl 58.44 g/mol; Molecular mass MgCl2 95.12 g/mol

    5.8 g NaCl (Final conc. 100 mM) 100 ml 1 M stock Tris, pH 9.5 (Final conc. 100 mM) 4.7 g MgCl2 (Final conc. 50 mM) 1.0 ml 100% Tween 20 (Final conc. 0.1%) Adjust to 1000 ml with MilliQ H20

    Filter the solution with 0.45 µm filter to avoid MgCl2 precipitation.

Contacts

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History

Created by bodil_oster on 2010-03-23.

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